A central concern of the present post-genomic era of biology is understanding the chemical and physical mechanisms by which gene expression is regulated. Appropriate activation and repression of particular genes is necessary for maintaining normal cell function and is required for executing the programs of cell differentiation that are essential to the development of multicellular organisms. Collectively, gene regulatory systems are the brain of the cell that allow it to respond appropriately to environmental stimuli. Many cancers and other diseases result from deranged gene regulation. In this research project, we have developed and begun applying an entirely new approach to studying the molecular mechanisms of transcription and transcription regulation in vitro. Instead of studying populations of molecules, we directly visualize the RNA polymerase and regulatory proteins on an isolated single DNA molecule, following the progression of the molecular machinery through its different states in real time while simultaneously observing the extent of transcriptional activation. Such direct visualization is made possible by a multi-wavelength single-molecule fluorescence approach we call CoSMoS (colocalization single- molecule spectroscopy). In this application, we propose applying the CoSMoS approach to elucidating the dynamic mechanisms of selected processes involved in regulation of transcription initiation and elongation using both prokaryotic and eukaryotic RNA polymerases and regulatory proteins in transcription reactions in vitro. Our goals are: 1) determine how secondary channel proteins GreB and DksA dynamically modulate the function of bacterial transcription elongation complexes; 2) elucidate the mechanism by which bacterial elongation complexes are loaded with and regulated by general elongation factors NusA and NusG; and 3) visualize eukaryotic RNA polymerase II initiation in nuclear extracts and from purified components, and characterize the dynamics of processivity factor recruitment and retention.